Use of Cerebral Open-Flow Microperfusion for the Longitudinal Collection of Interstitial Fluid in an Animal Model of Glioblastoma

We are characterizing soluble signaling components within the brain tumor interstitial fluid that contribute to disease progression and treatment response. Evaluating absolute solute concentrations within the brain tumor interstitial remains an investigational challenge requiring the use of sophisticated experimental methods. To begin, with a sterile scalpel, make a sagittal midline incision along the surface of the skull, starting between the eyes and extending caudally for 8 to 10 millimeters.

Then use sterile forceps to laterally retract the skin from the midline incision. Ensure both bregma and lambda are visible, and extend the incision if necessary. Next, with a sterile scalpel, incise the periosteum and retract the tissue laterally with forceps.

Apply 1%to 3%hydrogen peroxide with a sterile cotton-tipped applicator to clean the skull surface. Now use a 0.3 to 1.0 milliliter insulin syringe to apply 20 to 40 microliters of self-etching dental adhesive evenly onto the skull surface. Then expose it to a dental curing light to etch the adhesive.

Once cured, wipe away any excess adhesive with sterile cotton-tipped applicators and saline. Reposition the drill bit to the cOFM guide coordinate. Set the drill to 15, 000 revolutions per minute and drill a burr hole using the DV micromanipulator dial.

Frequently monitor skull thickness during drilling, and flush with physiological saline to remove debris and reduce heat. Repeat the drilling procedure at each anchor screw coordinate. Now install anchor screws using sterile forceps and a sterile screwdriver until inserted one to two thread lengths deep.

Then mount the cOFM guide and dummy assembly into the guide holder and attach the holder to the stereotactic frame. Position the guide tip above the burr hole and lower its slowly to the predetermined depth at 1 millimeter per minute. Apply the cementing agent to the skull in thin, uniform layers, covering areas under and around the cOFM guide and screws.

Use a dental curing light to harden each layer before applying the next one. Ensure the cement covers the entire area between components while avoiding the locking wedge. Detach the guide holder from the cOFM guide and slowly raise it using the DV knob of the micromanipulator.

Using forceps, remove the locking wedge from the cOFM guide and dummy assembly. Gently lift out the dummy from the guide. Use the ML and AP micromanipulator knobs to align the syringe infusion insert assembly over the cOFM guide.

Lower the infusion insert using the DV knob and secure it in position by firmly reinserting the locking wedge with forceps. Now inject 3 microliters of cell suspension from the syringe at 1 microliter per minute. Monitor the area around the cOFM guide to ensure that no cell suspension efflux is out of the assembly.

Use forceps to remove the locking wedge from the guide and infusion insert assembly. Then carefully lift the infusion insert from the guide using the DV adjustment knob. Pre-fill the push and pull tubing lines with artificial cerebrospinal fluid using gas-tight glass syringes.

Install the peristaltic section of the tubing into the pump head of the microperfusion pump. Connect the perfusate bag to the push tubing using a Luer lock connector. Then connect the sampling insert to the tubing using flanged connectors.

Attach the longer inlet shaft to the output end of the push tubing and the shorter outlet shaft to the input end of the pull tubing. Place the connected sampling insert into a sterile microcentrifuge tube containing 750 microliters of artificial cerebrospinal fluid. Connect the fraction collector needle to the pull tube output line using a flanged tubing connector.

Then perform a tubing flush for 20 minutes at 10 microliters per minute to eliminate air bubbles. Next, pre-fill the pull tubing line with artificial cerebrospinal fluid using gas-tight syringes. Reinstall the peristaltic tubing into the pump head of the microperfusion pump.

Connect the sampling insert again using flanged tubing connectors. Attach the inlet to the output end of the pull tubing and the outlet to the input end of the pull tubing. Place the insert into a sterile microcentrifuge tube with 750 microliters of artificial cerebrospinal fluid.

Perform another 20-minute tubing system flush at 10 microliters per minute to clear any residual air bubbles. Place an anesthetized animal in an appropriately sized jacket. Draw the forelimbs completely through the arm holes and fasten the jacket securely along the animal's back.

Use a pair of forceps to remove the locking wedge from the guide and dummy assembly. Then carefully lift the dummy insert out of the guide. Gently install the sampling insert into the guide and reinsert the locking wedge.

Remove the animal from anesthesia and place it into the rotating cage system. Attach the cage tether to the animal jacket and activate the rotating cage system. After cOFM sampling, deactivate the rotating cage system and detach the cage tether from the animal jacket.

Now use forceps to remove the locking wedge from the guide and sampling insert assembly. Carefully lift the sampling insert out of the guide, then gently insert the dummy insert back into the guide and reinsert the locking wedge. Detach the fasteners on the back of the animal jacket and gently remove the jacket from the animal.

Bioluminescent imaging confirmed the presence of tumors in animals with cOFM guide mediated tumor cell engraftment. Proteomic principal component analysis of cOFM-sampled interstitial fluid revealed distinct separation between temozolomide-treated and dimethyl sulfoxide-treated animal groups. Global metabolomic signal distributions overlapped almost perfectly between temozolomide and dimethyl sulfoxide samples.

Despite signal overlap, metabolomic volcano analysis revealed a distinct temozolomide-driven shift. Quantitative comparisons of metabolomic values from the same animals showed that recirculated configurations improved analyte concentrations while preserving relative metabolite proportions. Relative nutrient availability within the brain tumor microenvironment remains poorly understood.

This protocol is being used to address this gap. Characterizing nutrient availability and signaling within the tumor microenvironment can elucidate mechanisms of glioma progression and adaptive resistance to treatment.